This is a proposal to carry out a structure/function comparison of two different retrovirus integrases (INs) that highlight both similarities and differences in their respective viral and target DNA binding sites. The study will use a novel and highly predictive homo-tetramer model for HIV-1 IN with bound viral DNA to guide biochemical, biological, and molecular genetic studies. This model identified amino acids in close proximity to the first 15-16 base pairs of the viral DNA ends. These predictions were validated using an innovative strategy that substituted unique amino acid residues from structurally related positions in avian sarcoma virus (ASV) into HIV-1 IN. This produced IN variants that in many cases 3'process heterologous ASV to the detriment of homologous HIV-1 viral LTR DNA end substrates. Three of the variants represent sites where HIV-1 IN amino acid changes occur in the development of diketo acid drug resistance, lead compounds being tested or in use for treatment of AIDS. We now propose to complete mapping of viral and target DNA binding sites and test predictions of the model in the process of integration inside of cells. In Specific Aim 1, we will determine if mutations that disrupt LTR and/or target DNA binding detected using reconstituted systems cause defects to integration of viral into host DNA. For those that do, we will search for 2nd site revertants that restore IN activity and viral growth. We will also demonstrate that the selection of some combinations of amino acids that change in drug resistant HIV-1 integrases is related to affects on 3'processing. Finally, we will identify amino acid residues on ASV IN that recognize its viral DNA ends. This will determine if there is conservation of structure/function across different but functionally related INs. In Specific Aim 2, we will identify amino acids on the IN surface that define the target DNA binding site using mutagenesis, drug sensitivity, and photo-cross linking techniques. Taken together, these innovative studies will provide a new understanding of the mechanism of retrovirus DNA integration and provide a vetted structural model that can be used to examine IN/host protein complexes, for drug design, and future development of enzymes capable of site specific integration. PUBLIC HEALTH RELEVANCE: These innovative studies will add significantly to our knowledge of the mechanism of retrovirus catalyzed DNA integration. They impact on the design of inhibitors directed towards IN and identify amino acid residues likely to be substituted in drug resistant enzymes and why.